Hazard-free microencapsulation for structurally delicate agents, an application of stable aqueous-aqueous emulsion

ABSTRACT

This invention provides method for sustained release delivery of structurally delicate agents such as proteins and peptides. Using unique emulsion system (Stable polymer aqueous-aqueous emulsion), proteins and peptides can be microencapsulated in polysacchride glassy particles under a condition free of any chemical or physical hazard such as organic solvents, strong interfacial tension, strong shears, elevated temperature, large amount of surfactants, and cross-linking agents. Proteins loaded in these glassy particles showed strong resistance to organic solvents, prolonged activity in hydrated state, and an excellent sustained release profile with minimal burst and incomplete release when being further loaded in degradable polymer microspheres. This invention provides a simple yet effective approach to address all the technical challenges raised in sustained release delivery of proteins.

CROSS REFERENCE OF RELATED APPLICATION

This application claims priority of U.S. Ser. No. 60/384,971, filed Jun.3, 2002, and U.S. Ser. No. 60/418,100, filed Oct. 11, 2002, the contentsof which are incorporated by reference here into this application.

Throughout this application, various references are referred to.Disclosures of these publications in their entireties are herebyincorporated by reference into this application to more fully describethe state of the art to which this invention pertains.

FIELD OF THE INVENTION

The present invention demonstrates a novel method for preparing a novelparticulate glassy system which effectively preserve structure/activityof proteins peptides, DNA, liposomes and live viruses during formulationprocess, storage, and application.

BACKGROUND OF THE INVENTION

Due to the impermeability and short half-life, most of proteintherapeutics require frequent injection. To reduce injection frequency,development of sustained release dosage forms has been a long-standingresearch focus since 1970s (1). In spite of extensive research efforts(2), up to now, sustained release formulation technology has succeededin only one protein drug, recombinant human growth hormone (rhGH). Themajor roadblocks are invariably the protein instability in formulationprocess and at the site of release (3, 4) as well initial burst andincomplete release.

Various strategies to improve protein stability in microencapsulationhave been reported in last decades (3, 5, 6). Many of these approaches,however, only address one or some issues, leaving others unsolved oreven creating new problems. Some methods are feasible for only specificproteins, and some reports are contradictory to each other due todifferent focal points of researchers. For example, for the onlycommercially available long-acting protein, sustained release rhGH, theprotein was stabilized by forming complex with zinc ions (7) based onthat natural hGH forms complex with zinc in secretory granules (8). Whenzinc was co-encapsulated with another protein, erythropoietin (EPO) forexample, up to 40% of released proteins was aggregated (9), which couldresult in unwanted immunogenisity. In order to protect proteins fromorganic solvents used in microencapsulation, sugars, inorganic salts orother conceivable excipients are used to preformulate proteins intosolid particles prior to microencapsulating them into degradable polymermicrospheres through a solid-in-oil-water (S-O-W) emulsification process(7, 9, 10). These excipients often resulted in considerable burstrelease due to strong osmotic pressure created by their high solubility(11) and rapid dissolution (12). When highly soluble ammonium sulfatewas used to stabilize EPO in microencapsulation, burst release accountedup to 55% of total drug (9).

Cleland and Jones studied the effects of various excipients onprotection of rhGH and interferon (IFN-) in water-in-oil-in-water(W-O-W) and S-O-W encapsulation processes, and found that mannitol ortrehalose were the best in preventing proteins from aggregation duringmicroencapsulation process were prevented (6). Sanchez et al. examinedthe protection effects of similar excipients for another protein,tetanus toxoid, and found dextran, that was ineffective for recoveringrhGH and IFN-γ in Cleland and Jones report, showed best protection forthe protein (based on ELISA) at the release phase under a hydratedcondition (10). It seems that small sugars offer better protection indehydration steps (drying), while polysaccharides are more effective ina hydrated step (release) (13). A burst release of 60% of total loadingwas observed from dextran included PLGA microspheres prepared by Sanchezet al. This burst release may be attributed to the particle size of theco-lyophilized protein-excipient particles (14, 15).

The size of pre-formed protein particles plays an important role in aS-O-W process. Morita et al. demonstrated that when the mean diameter ofsolid protein particles increased from 5 to 20 μm, the initial releasealmost doubled, and encapsulation efficiency dropped from 80% to 20%(15). Cleland et al. discussed different approaches for reducing proteinparticle size for a S-O-W process (6). Homogenizing a lyophilizedprotein-excipients powder in organic solvents can only result particlesabove 10 μm in diameter, while milling the powders to smaller size maycause protein denature due to the shears and heat generated (6). Spraydrying may produce protein particles to desired size, but shear and heatat atomization as well as the presence of air-liquid interface may causedenaturation (6, 16). Moreover, surfactants must be used in spray dryingand spray freeze-drying that facilitate contact and interaction betweenproteins and dichloromethane (the solvent most frequently used inmicroencapsulation) (6). Maa et al. reported that complexation of rhGHwith zinc prior to spray drying can effectively prevent aggregation ofthe protein (16). Again, zinc complexation can denatrue proteins otherthan rhGH (9). Morita et al. prepared fine protein particles by afreezing-induced precipitation with a co-solution of proteins and PEG(15, 17). But the protein particles still have to be exposed to organicsolvents directly during microencapsulation. Direct contact ofunprotected proteins with PLGA will cause incomplete release by strongadsorption of the proteins on the internal surface of the polymer matrix(18). To avoid the hydrophilic-hydrophobic interface, aqueous two-phasesystems were used for preparing polysaccharide particles (19, 20).However, the dispersed phases need to be stabilized by covalent or ioniccross-linking, another potential cause for protein denaturation.

For sustained release of delicate proteins, an approach that can addressall these important issues is highly desired. Due to the long-standingdifficulties discussed above, it is unlikely that this task can beaccomplished with the existing approaches. Microencapsulation strategiesbased on new scientific concepts are required.

In one of our previous patent application, we have reported (as thefirst time according to best of our knowledge) a uniquemicroencapsulation system, stable polymer aqueous-aqueous emulsion (24).This system differs from conventional emulsions in that both thedispersed and continuous phases are aqueous. The system is alsodifferent from so-called polymer aqueous two-phase systems that form twoblock phases in absence of continuous mixing. This emulsion is stablefor up a week without any (covalent or ionic) cross-linking treatment.Due to these unique characteristics, delicate therapeutics such asproteins, liposomes or live viruses can be loaded into the droplets ofthis emulsion under a condition free of chemical or physical hazardssuch as organic solvents, concentrated salts, extreme pH, crosslinkagents, high shear stress, high interfacial tension and hightemperature. By freeze-drying or other drying methods, dispersed phaseof the emulsion can form glassy particles of defined shape and uniformsize for various delivery purposes (inhalation or sustained release).Our previous work has established the proof-of-principle that all thestability problems raised in protein microencapsulation, such as theprocesses of protein loading, drying, storage and release (3), can beaddressed using this unique system. In addition, all the ingredientsused are those proven for injection into human.

This present application further demonstrates applications of thisstable aqueous-aqueous emulsion system in delivery of protein drugs.Proteins can be loaded into the dispersed phase of the aqueous-aqueousemulsion system and form glassy particles by freeze-drying thereafter.The entire preparation process is free of any chemical physical hazards.Protein activity can be well preserved during this preparation process.Proteins loaded in the glassy particles made via the emulsion system(called AqueSphere(s) hereafter) showed strong resistance to organicsolvents, prolonged activity in hydrated state at 37° C., as well aslinear release profile with minimal burst and incomplete release whenbeing further loaded in degradable polymer microsphere.

SUMMARY OF THE INVENTION

It is an object of this invention to provide a method to preparepolymeric microspheres for sustained release of protein therapeutics.The method is an application of material system, stable polymeraqueous-aqueous emulsion and AqueSphres (polysaccharide glassy particlesmade by solidification of the emulsion system), which were described inour earlier patent application (24). The method comprises 1) loadingproteins in the droplets of the stable aqueous-aqueous emulsion system;2) preparation of AqueSpheres with diameter ranging between 1-5 micronsfor inhalation protein delivery; 3) encapsulation of AqueSpheres intoPLGA and other degradable polymer microspheres and injectable implants;4) preparation of AqueSpheres loaded with structurally delicatesubstances other than proteins (such as liposomes and live viruses) forinhalation, nasal spray and other therapeutic uses.

A major difficulty that delayed development of sustained release ornon-invasive protein formulations is that proteins are denatured duringthe formulation process. To prevent protein denature, a formulationprocess must be free of (or proteins must be protected from) thechemical physical hazards discussed above. In achieving this objective,however, properties and functions of the final product such as particlesize and shape, release profile, loading efficiency, prolonged activityat the site of release and so forth should not be compromised. It isalso preferred that the manufacture process can be easy, reproducibleand environmentally friendly.

The present invention has demonstrated a simple solution for all theseobjectives above.

First, fragile biological agents such as proteins can be loaded into thedispersed phase of the stable polymer aqueous-aqueous emulsion system(24) under a condition free of any chemical or physical hazard. Auniform size distribution of the droplets can be achieved by aconventional emulsification process under appropriate shear stress andlow interfacial tension (due to the aqueous-aqueous nature). Then thesystem can be freeze-dried to dry powder in which the polymer dropletsconverted to glassy particles of uniform sizes (1-5 um in diameter).Once the glassy particles are formed, the structure of the loaded arepreserved and protected. Due to its hydrophilicity and high glassytransition temperature, the system offers strong resistance to organicsolvents as well as resistance to ambient temperature and moisture (interms of protein activity retention). The bio-agents-loaded AqueSpherescan therefore be used for inhalation drug delivery (based on their sizerange) or subjected to further formulation process with biodegradablehydrophobic polymers for sustained release.

For preparation of sustained release microspheres, AqueSpheres can beloaded into PLGA (or other degradable polymers) microspheres byconventional solid-in-oil-in-water (S-O-W) or solid-in-oil-in-oil(S-O-O) emulsification methods. A recovery experiment from PLGAmicrospheres indicated that the AqueSpheres remain intact inside of themicrospheres (Example 4).

Bioactivity of the proteins loaded in AqueSpheres was retained aftercontacted with organic solvents and after microencapsulation process asassayed in cell proliferation (Example 5, 6, and 7), indicating thatconformation of proteins were well protected in the glassy matrix ofpolysaccharide. In addition, the activity retention of proteins aftermiroencapsuleted in PLGA microspheres (Example 7) suggests highencapsulation efficiency.

The most challenging task in developing sustained release protein dosageforms is to ensure protein activity in a hydrated state at physiologicaltemperature (21). Hydration and temperature elevation will increase themobility of proteins and lower the energy barrier for proteinhydrolysis, aggregation and conformation change. With the presenttechnology, proteins loaded in AqueSpheres showed prolonged activity ina hydrated state at 37° C. (Example 8). Recombinant human erythropoietin(rhEPO) which has in vivo half life of 8.5 hrs and in vitro half life ofa day showed a half life of a week under a hydrated condition whenloaded in AqueSpheres (Example 8). The AqueSphere matrix formed aviscous phase surrounding the proteins so that limited protein mobilityand the chance for proteins to contact with each other and other species(the degradable polymer and enzymes).

Burst effect, defined as rapid release of considerable amount ofloadings in the initial period of administration, is another commonproblem in developing sustained release dosage forms of protein drugs.Burst effect is found for both injectable implants and microsphereformulations, although the causes may be different. Accompanying withburst effect is incomplete release that part of the proteins loadedstrongly interact with the polymer matrix and are not able to release inthe required period. Having proteins pre-encapsulated in AqueSpheresprior to loading into degradable polymers can effectively prevent bursteffect, and at the same time, reduce the portion of incomplete release(Example 9).

Moreover, AqueSpheres helps to reduce local acidity generated by polymerdegradation. Local acidity is another cause believed for proteindenaturation during release period. AqueSpheres form inter-connectedchannels when being hydrated in degradable polymer matrix that theirviscous nature limits diffusion of macromolecular proteins but permeableto small molecular buffers. This nature allow the local acidity bebuffered in the sustained release process. In addition, the surfacemodifier (sodium alginate) itself possesses significant buffer effect.

This invention provides a simple yet effective solution for all thelong-standing technical difficulties in developing sustained releaseprotein microspheres (3-5).

DETAILED DESCRIPTION OF THE FIGURES

FIG. 1. Stable polymer aqueous-aqueous emulsion loaded with myoglobin inthe dispersed phase. The picture was taken one week after the sampleswere prepared.

(1) Dispersed phase: 1 ml, containing 5 w/w % myoglobin and 20 w/w %dextran; Continuous phase: 5 ml, containing 1 w/w % sodium alginate and20 w/w % PEG.

(2) Dispersed phase: 1 ml, containing 5 w/w % myoglobin and 20 w/w %dextran; Continuous phase: 10 ml, containing 1 w/w % sodium alginate and20 w/w % PEG.

(3) Dispersed phase: 0.5 ml, containing 5 w/w % myoglobin and 20 w/w %dextran; Continuous phase: 10 ml, containing 1 w/w % sodium alginate and20 w/w % PEG.

(4) Dispersed phase: 1 ml, containing 5 w/w % myoglobin and 20 w/w %dextran; Continuous phase: 5 ml, containing 20 w/w % PEG.

(5) Dispersed phase: 1 ml, containing 5 w/w % myoglobin and 20 w/w %dextran; Continuous phase: 5 ml, containing 1 w/w % sodium alginate, 20w/w % PEG and 10 mM NaCl.

(6) Dispersed phase: 1 ml, containing 5 w/w % myoglobin and 20 w/w %dextran; Continuous phase: 5 ml, containing 1 w/w % sodium alginate, 20w/w % PEG and 100 mM NaCl.

The brown dispersed phase (myoglobin/dextran) in samples (4) and (6)started to fuse right after preparation and formed a block phases at thebottom of over night. Those in sample (1), (2), (3) and (5) wereunchanged in a week as observed using a microscope.

FIG. 2. Microscopic images of stable aqueous-aqueous emulsion andpolysacchride particles.

(2A) Microscopic image of the stable aqueous-aqueous emulsion shown inFIG. 1-1; (2B) microscopic image after (2A) was freeze-dried and washedwith dichloromethane (to remove the dried PEG phase

FIG. 3. Preparation of polylactic-glycolic acid (PLGA) microspheres by aS-O-W double emulsification

3A) Microscopic image of a S-O-W double emulsion for which AqueSpheresare evenly suspended in the organic PLGA phase.

3B) Solidified PLGA microspheres in which AqueSpheres are encapsulated.

FIG. 4. Microscopic image of AqueSpheres recoved from PLGA microspheres(as shown in FIG. 3B). The size and shape of recovered AqueSpheres areidentical to that before encapsulated in PLGA microspheres (FIG. 2B).

FIG. 5. Comparation of catalytic activity of β-galactosidase assayed ateach step of microencapsulation using AqueSphere technology.

Compared with β-galactosidase loaded in a fresh aqueous-aqueousemulsion, its activity only slightly reduced in subsequent steps.

FIG. 6. Bioactivity of rhEPO assayed by proliferation of TF1 cells aftereach preparation step.

Equivalent amounts of rhEPO were reconsitituted and incubated with TF1cells after emulsification, freeze-drying, and washing withdichloromethane, respectively. Cells proliferated were counted under amicroscope. Numbers of cells per well were averaged from three wells.

FIG. 7. Bioactivity of recombinant human granulocyte macrophage colonystimulating factor (rhGM-CSF) assayed by proliferation of TF1 cellsafter each preparation step.

Equivalent amounts of rhGM-CSF were reconsitituted and incubated withTF1 cells after emulsification, freeze-drying, washing withdichloromethane, and recovery from PLGA microspheres in which theprotein was encapsulated, respectively. Cells proliferated were countedunder a microscope. Numbers of cells per well were averaged from threewells.

FIG. 8. Bioactivity of rhEPO assayed by proliferation of TF1 cells afterincubation in a hydrated form at 37° C. Activity after incubattion in ahydrated state at physiological temperature: The protein loaded inAqueSpheres was added with water twice of their mass and incubated 37°C. for different days prior to cell culture. The activity was indicatedby the average number of cells grew in each well. For control,equivalent amount of rhEPO was incubated in a PBS buffer and assayedunder identical conditions.

FIG. 9. Bioactivity of rhGM-CSF assayed by proliferation of TF1 cellsafter incubation in a hydrated form at 37° C. Activity after incubattionin a hydrated state at physiological temperature: The protein loaded inAqueSpheres was added with water twice of their mass and incubated 37°C. for different days prior to cell culture. The activity was indicatedby the average number of cells grew in each well. For control,equivalent amount of rhGM-CSF was incubated in a PBS buffer and assayedunder identical conditions.

FIG. 10. Catalytic activity of AqueSphere-loaded β-galactisidase as afunction of incubation time in a hydrated state at 37° C. The activitywas compared with that incubated in a trehalose solution. Concentrationof sugars (or polysaccharide) was 30 w/w % in both hydrated AqueSpheresand trehalose.

FIG. 11. Release profile of myoglobin from PLGA microspheres. Therelease study was carried out by suspending 50 mg microspheres in 2 mlof 0.1 M BPS buffer at 37° C. Amount of myoglobin released was assayedusing a BCA method. ♦: Pure myoglobin particles directly encapsulated inmicrospheres made of ester-end PLGA with lactide/glycolide ratio of50/50 and molecular weight of 6K; ⋄: Myoglobin-dextran particlesencapsulated in microspheres made of the same PLGA as above.

FIG. 12. Release profiles of myoglobin microencapsulated in PLGAmicrospheres as AqueSpheres. ◯: from microspheres of PLGA withlactide/glycolide ratio (L/G) of 50/50 and molecular weight (MW) of 12K;□: from microspheres of PLGA with L/G of 65/35 and MW of 12K; Δ: frommicrospheres of PLGA with L/G of 75/25 and MW of 12K; ▪: frommicrospheres of PLGA with L/G of 65/35 and MW of 20K.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method to use polymer aqueous-aqueousemulsion system [24] to deliver proteins and other biological agents ina sustained release dosage forms. When biological agents are loaded in apolysaccharide solution, followed by emulsification and freeze-dry,their structure is “fixed” in a hydrophilic glassy matrix. Such glassyparticles (AqueSpheres) offer a series advantages that cannot be allachieved by any other reported method.

Small and uniform particle sizes of pre-protected proteins play animportant role in control of the burst release and improvingencapsulation efficiency in a S-O-W or a S-O-O micronecapsulationprocess (6, 13). This invention provides a method to prepareprotein-loaded polysaccharide glassy particles of defined shape anduniform size (1-3 um, Examples 1 and 2) under a condition free oforganic solvents, strong interfacial tension, strong shears, elevatedtemperature, large amount of surfactants, and (covalent or ionic)cross-linking agents. These factors are known to denature proteins inone or several steps of microencapsulation process (3,6,21). Asdiscussed above, however, no a method known to date (W/O emulsion, spraydrying, spray freeze-drying, freeze-drying, milling, and in situcross-linking) can be used for preparing protein particles withoutcompromising with the hazards above.

In addition, spray drying and spray freeze-drying can only be used toprepare particles with low molecular weight sugars or salts as theprotein stabilizers because polysaccharide solutions are too viscous tospray. The present stabilized emulsification method allows viscousaqueous solution be easily dispersed. As discussed later, polysaccharidestabilizers possesses a number of advantages for both proteinstabilization and release kinetics.

Once loaded in the polysaccharide particles, delicate proteins can beprotected from contact with organic solvents during microencapsulationprocesses. β-galactosidase, recombinant human ethrypoietin (rhEPO) andrecombinant human granulocyte macrophage colony stimulating factor(rhGM-CSF) were loaded in AqueSpheres and washed with dichloromethane(DCM) and/or encapsulated in PLGA microspheres with DCM as the solvent.The bioactivity of these proteins can be well retained as determinedwith activity assay after the preparation treatments (Examples 5, 6, and7). Contact with organic solvents is believed as the major chemicalhazards in microencapsulation processes using degradable polymers (3).

In addition to resistance to organic solvents, AqueSpheres can protectproteins from aggregation and conformation change in a hydrated state atphysiological temperature. Protecting delicate protein under such acondition is regarded as the most challenging technical hurdle insustained protein release (21). We incubated hydrated-AqueSphres, loadedwith rhEPO, rhGM-CSF and β-galactosidase respectively, at 37° C., andfound that protein activity were well retained (Examples 8). For rhEPO,its half-life in hydrated AqueSpheres was 7 times longer than that in aBPS buffer (FIG. 8 and Example 8). For rhGM-CSF, there was nosignificant declining in bioactivity after incubation for 9 days (FIG.9, and Example 8). For β-galactosidase, a comparison was made betweenAqueSphres and trehalose (a well recommended protein stabilizer (6)matrix under the same incubation condition. Proteins protected byAqueSpheres were 5 times active as that protected by trehalose afterincubation for a week (FIG. 10 and Example 8).

AqueSpheres, when encapsulated in degradable polymer microspheres, offeran ideal release profile with extended linear kinetics and free ofburst. Polylactic-glycolic acid (PLGA) microspheres are know to releaseloaded macromolecules in three phases (22): an initial burst due torapid diffusion of the molecules located in the surface region (25) orinternal water-filled pores (14) of the microspheres, a lag phase afterthe initial burst, and an accelerated release due to bulky degradationof the polymer. A burst effect, for which more than 50% loading may bereleased in the first day after administration, may be dangerous formany therapeutic agents. Due to the small and uniform size, particlesprepared by this method dispersed evenly in the matrix of degradablepolymers (Example 3) that there is no a surface-rich proteindistribution. In addition, unlike small molecular weight proteinstabilizers that readily dissolve (cause high osmotic pressure (11)) andrapidly diffuse out of the polymer matrix, AqueSpheres form a viscousphase that fills the diffusion channels when hydrated. Since themolecules of polysaccharide themselves diffuse gradually from thepolymer matrix (23), protein burst can be suppressed (Example 9) by theviscous stabilizers. Moreover, the diffusion process may be extended sothat it overlaps with the degradation process to give a single phaserelease kinetics (Example 9).

Interaction between proteins loaded and the degradable polymers isanother problem that causes incomplete release and insoluble proteinaggregation (18). In the present method, the protein molecules aresurrounded by the viscous polysaccharides in side of a hydratedmicrosphere during the entire release period (23) so that the chance forprotein-polymer contact is reduced. Release profiles of myoglobinencapsulated in PLGA microspheres directly and the encapsulated throughAqueSpheres are compared in FIG. 11 (Example 9). For directmicroencapsulation, less than 20% of the loaded proteins were releaseover 45 days. While for encapsulation through AqueSpheres, 70% of theloadings were released for the same period.

Local acidity in the PLGA matrix is another cause for protein denature(3). When the polymer degrades, the degradation products (lactic acidand glycolic acid as well as their oligomers) may be trapped inside ofthe polymer matrix and cause the local pH to decrease. In our system,AqueSpheres form an interconnected viscous phase when hydrated. Theseviscous channels, although less permeable to macromolecular proteins,are permeable to small molecular buffers so that the acidity degradationfragments may be buffered. In addition, alginate used as the surfacemodifier for the aqueous-aqueous emulsion (example 1) possesses a buffereffect. In a titration test, the pH was stabilized around 5 when 100 μlof 0.1 N HCl was added to 0.9 ml 150 mM (based on the monomer) alginatesolution. For same amount of water, 10 μl of the same acid caused pH todrop to 1.

The present invention provides, as the first time, a simple yetinclusive solution by which all the technical challenges in sustainedrelease protein delivery can be addressed. With this method, delicateproteins can be protected in steps of both formulation andadministration, and release approximately constantly with minimal burstand incomplete release. The system demonstrated is expected to have awide variety of applications for delivery of delicate therapeutics.

The invention will be better understood by reference to the Exampleswhich follow, but those skilled in the art will readily appreciate thatthe specific examples are only illustrative and are not meant to limitthe invention as described herein, which is defined by the claims whichfollow thereafter.

EXAMPLES Example 1 Stability of Polymer Aqueous-aqueous Emulsion

Stability of polymer aqueous-aqueous emulsion was examined byobservation of the fusion (the size change) of the dispersed phase undera microscope and by observation of formation of block phases of thecolored dispersed phase directly by eyes as a function of time. Thedispersed phase was formed by a dextran solution. Three concentrationsof the dextran solution, 5, 20 and 40 w/w %, were used in theexperiments without significant difference in the results, i.e. foreither of the concentration, stable aqueous-aqueous emulsion was formed.For the average molecular weight of dextran, <M>_(W)=10,000, 67,000 and500,000 were tested without significant difference in results. Thecontinuous phase contained PEG with concentration 5, 20, and 40 w/w % indifferent tests, for all of which, stable emulsion was formed. Averagemolecular weight of PEG used were 8000 and 22,000. As emulsionstabilizers, sodium alginate, carboxymethyl dextran, carboxymethylcellulose were tested. All these stabilizing agents showed effectivenessin stabilizing the aqueous-aqueous emulsion. Sodium alginate (<M>_(W)was represented by low, medium or high viscosity) was used in most ofexperiments for its abundant sources. The concentration of the emulsionstabilizers, 0.2, 1, 5 w/w %, were used in experiments, respectively.The emulsion stabilizers were co-dissolved with the dispersed phase andthe continuous phase, respectively. No significant difference inemulsion stability was observed. For direct observeation, coloredmolecules, blue dextran (<M>_(W)=50,000 and 1,000,000) or myoglobin wasadded into the dispersed phase as an indicator.

Emulsions with various concentrations of sodium chloride were preparedby adding the dextran solution to the PEG solution, followed byhomogenizing with a mechanic homogenizer. Dextran to PEG ratio was 1:5to 1:20. After the emulsions were prepared, a drop of the sample wassubjected to a microscope for microscopic image taking. Then the sampleswere left in bottles for continuous observation.

FIG. 1 shows a picture of a polymer aqueous-aqueous emulsion aftermixing was stopped and the emulsion stored at room temperature for aweek. Myoglobin was used as a model protein that was loaded in thedispersed phase, showing the rusty color. Among the six samples, sample4 was prepared without sodium alginate. Sample 6 was the same as samples1 except that sodium chloride was added (to reach 100 mM). For these twosamples, fusion of the dispersed phase occurred right after stirring wasstopped, which led to formation of two block phases in a few hours. Forthe other four samples in FIG. 1, the droplet diameter remained in therange of 3-7 μm (FIG. 2 A) during the week. This result supported ourhypothesis that charged polymer molecules adsorbed at the dropletsurface and created a diffuse double layer. Increasing the concentrationof sodium ions, the counter ions of alginate, shelled the surfacecharge, reduced the magnitude of the zeta potential, and thus causeddroplets to coalescence. Reducing the dextran/PEG ratio to 1:15 lead toan emulsion stable for two weeks.

In this experiment, the partition coefficient of myoglobin between thecontinuous phase and the dispersed phase was 1:50, as determined byabsorbance at 410 nm, indicating that the majority of myoglobin was inthe dextran phase. In addition to myoglobin, recombinant humangranulocyte macrophage colony stimulating factor (rhGM-CSF) andliposomes carrying amphotericin B (AmB) were also loaded in the systemand formed glassy particles similar to those in FIG. 2B. About 93% ofrhGM-CSF and 95% of AmB/liposomes were partitioned in the dispersedphase as detected by activity assay (See later discussion) and UVabsorbance at 408 nm, respectively.

FIG. 2A shows a microscopic image of a polymer aqueous-aqueous emulsionprepared with the sodium chloride free solutions described above.Emulsion droplets with a uniform size distribution between 3-8 micronsin diameter were obtained.

Example 2 Preparation of AqueSpheres

AqueSpheres were prepared simply by freeze-drying the stable emulsionsof above. After freeze-drying, the dextran droplets converted to solidparticles. However, the most of dextran particles were dispersed in asolid matrix formed by the continuous phase, PEG. The PEG can be removedby washing the lyophilized powder with methylene chloride oracetonitrile. These solvents did neither dissolve nor swell the drieddextran phase. FIGS. 2A and 2B showed the microscopic images of thedispersed phase at different preparation stages: after emulsification,after freeze-drying followed by rinsing with dichloromethane (to removePEG), and after recovery from PLGA coating, respectively. Afterfreeze-drying, the diameter of the dispersed phase remained uniform butdropped from 3-7 μm to 1-3 μm, a reasonable size reduction from loss ofwater (See FIG. 2B). These images indicated that no droplet fusionoccurred during lyophilization. This size range of the dried particles(1-3 μm) is ideal for inhalation delivery of therapeutics and is alsosuitable for preparation of degradable polymer coated microspheres viadouble encapsulation (S-O-W) (5, 13).

Example 3 Microencapsulation of AqueSpheres into PLGA Microspheres

AqueSpheres can be further microencapsulated into the matrix of PLGA andother biodegradable polymer microspheres through a“solid-in-oil-in-water” emulsification process. In the present study,PLGA with lactic:glycolic ratio of 50:50 and 75:25 were used.AqueSpheres prepared as in Example 2 were first suspended in aPLGA/dichloromethane solution (10-20%) at the AqueSphere/PLGA ratio of1:2 to 1:20, then added into a water solution containing 0.1-10% sodiumchloride and 0.1-4% polyvinyl alcohol (PVA) or PEG or polyvinylparralidone (PVP) under stirring. The volume ratio of the two solutionswas 1:2 to 1:10. After an emulsion was formed, the organic solvent wasextracted by pouring the system into large volume of cold water (10times of the emulsion) under stirring. FIGS. 3A and 3B show themicroscopic images of the PLGA droplets before solvent extraction andPLGA particles after solvent extraction, respectively. Before solventextraction, the PLGA droplets were transparent within which theencapsulated AqueSpheres were evenly distributed. After hardening bysolvents removing, the PLGA particles lost transparency.

Example 4 Recovery of AqueSpheres from PLGA Particles

AqueSpheres can be recovered from the PLGA microspheres prepared as inExample 3. AqueSpheres loaded PLGA particles were re-dissolved indichloromethane or acetonitrile, followed by centrifugation. Thisprocedure was repeated 4 to 6 times. FIG. 4 shows the AqueSpheresrecovered from PLGA microspheres by the above mentioned procedure. Theparticle size and shape of AqueSpheres remain the same as before beingencapsulated in PLGA microspheres. The result suggests that hydration ofAqueSpheres during the microencapsulation process is not significant.

A weight measurement was carried out to examine encapsulation efficiencyof AqueSpheres by the PLGA microspheres. A relatively constant weightratio of dextran to PLGA was obtained before (1:19) and after (1.06:19)microencapsulation, suggesting high encapsulation efficiency. Thisconclusion consists with our result on protein activity assay before andafter encapsulation (See Example 7).

Example 5 Protection of β-galactosidase by AqueSpheres Against OrganicSolvents

To examine the effectiveness of AqueSpheres in protecting delicateproteins against organic solvents, β-galactosidase, an enzyme withquadral structure and molecular weight of 434 KD, was loaded intoAqueSpheres. The protein was dissolved in a dextran solution (MW=10-500KD, 5-25% in concentration) at the ratio of 10-100 units/ml andemulsified into a PEG solution as in Example 1. After freeze-dying, thePEG phase was removed by washing with dichloromethane (a popular solventused in preparation of PLGA microspheres) several times as in Example 4.Then, the obtained protein-loaded AqueSpheres were re-dissolved in abuffer and assayed by hydrolysis of o-nitrophenyl-β-D-galactopyranoside(ONPG). As indicated in FIG. 5, the catalytic activity of the enzymeonly decreased less than 10% after the procedure from Example 1 throughExample 2 (included emulsification, freeze-drying and washing withdichloromethane). The result was reproducible by three runs. This 10%activity loss includes loss of the proteins by partition between thedextran and PEG phases in the emulsification process and by the washingprocess, as well as those denatured in freeze-drying and in the washingprocess and lost during the washing process. This result indicates thatdelicate proteins inside of AqueSperes can be well protected againstorganic solvents during microencapsulation process.

Example 6 Partition of rhEPO and rhGM-CSF in the Dispersed and theContinuous Phases of the Aqueous-aqueous Emulsion

A partition experiment was carried out to determine the efficiency ofproteins being loaded in the dispersed phase of the emulsion system. Theaqueous-aqueous emulsion containing recombinant human erythropoietin(rhEPO) or recombinant human granulocyte-macrophage colony stimulatingfactor (rhGM-CSF) was centrifuged, followed by a cell proliferationassay using a TF1 cell line. Protein activity was measured by countingthe numbers of cells per well under a microscope. About 94% of rhEPO and93% of rhGM-CSf were found in the dextran phase by the partitionexperiment.

Example 7 Protection of rhEPO and rhGM-CSF by AqueSpheres AgainstOrganic Solvents

Protein-protection by AqueSpheres was further examined with the twoproteins rhEPO and rhGM-CSF. The proteins were loaded in AqueSpheres andtreated according the procedure identical to that in Example 5.Bioactivity of the proteins was assayed by the same cell proliferationmethod as for partition (Example 6). The proteins before encapsulationand recovered from AqueSpheres (after washing with dichloromethane) wereadded into same cell suspensions, respectively. The result for rhEPO isshown in FIG. 6. After freeze-drying, the activity retention for rhEPOwas 85% as indicated by the drop of cell count from 27800 to 23700 perwell. Washing the lyophilized powder (so the Peg phase was removed)resulted a further drop of the cell count to 22600, indicating that theactivity retention was 95%. Because only 94% of proteins were remainedin the dextran phase after washing with organic solvent (Example 6), theactivity retention was 100% after contact with the organic solvent.

FIG. 7 shows the result of activity assay for rhGM-CSf after eachpreparation step. Freeze-drying the protein-loaded emulsion to a drypowder caused the average number of cells per well slightly reduced from130900 to 122600, indicating roughly 94% of activity retention. Afterwashing the freeze-dried powder with dichloromethane to remove residualPEG, the cell count decreased to 111100 per well, a 9% furtherreduction. Much of this 9% reduction, however, was caused by rhGM-CSFpartitioned in the continuous phase (about 7% of total rhGM-CSF, Example6) that was washed away along with PEG. Encapsulating the protein-loadeddextran particles into PLGA microspheres did not cause further activitydecrease as indicated by an average cell count of 118900 per well. Thehigh activity retention also indicated high encapsulation efficiencythat was indicate by a weight measurement (Example 4).

Example 8 Activity Retention of rhEPO, rhGM-CSF and β-galactosidase byAqueSpheres in Hydrated State at Physiological Temperature

It has been widely believed that the most challenging task in developingsustained release dosage forms of protein drugs is to ensure proteinactivity in a hydrated state at physiological temperature (18). Duringsustained release, the degradable polymer microspheres will absorb waterand swell, and the encapsulated protein molecules will be exposed to ahydrated condition at body temperature. Hydration and temperatureelevation will increase the mobility of protein molecules that increasesthe chance for chemical or physical changes of protein (19). To examineprotein stability under physiological conditions, water was added to thedextran particles loaded with rhEPO or rhGM-CSF (to formed a viscous 30w/w % dextran solution) and incubated at 37° C. Protein activity in FT1cell proliferation was shown in FIGS. 8 and 9 as a function ofincubation time.

For rhEPO, activity of those protected by AqueSpheres gradually declinedto about 50% in a week (FIG. 80). For unprotected rhEPO, however, thesame amount of activity declining took only one day. Half-life of rhEPOis 8.5 hrs in vivo due to enzymatic catalysis in the body. Clearly theviscous polysaccharide phase, formed by hydration of AqueSphere, canextend the protein activity at physiological condition for significantperiod of time.

Similar result was obtained for rhGM-CSF (FIG. 9). For protectedrhGM-CSF, activity retention was 85% after 10 days of incubation. Thatof unprotected VhGM-CSF was 56% for the same incubation period.

The protection effect of polysaccharide stabilizers for β-galactosidasein hydrated state was compared with that of trehalose. The activityassessment was carried out same as in Example 5. After 7 days ofincubaion at 37° C., the activity for the protein stabilized bypolysaccharide declined to 89% while that stabilized by trehalosedeclined to 17%. Extending the incubation time to two weeks resulted ina further activity reduction to 48% for hydrated AqueSpheres but 0% forthat incubated in trehalose solution.

Example 9 Protein Release Profile with Mimimal Burst and IncompleteRelease from PLGA Microspheres

Burst effect and incomplete release are another common problem indevelopment of sustained release dosage form of protein drugs. Due toburst effect, 30-70% of proteins loaded maybe release immediately afteradministration. Incomplete release referes to that 20-40% of theloadings remained as insoluble residues. This undesired release can beprevented by pre-loading proteins in AqueSphere. The protein was loadedinto AqueSpheres (0.1-20%) through the aqueous-aqueous emulsificationprocess first. Then the protein-loaded AqueSpheres were encapsulated inPLGA microspheres (1-20%) using a S-O-W technique. Loading capacity ofmyoglobin in PLGA was 0.25 to 5%. PVA, PEG and PVP were dissolved in thewater phase (0.1-5%) as surfactants. FIG. 11 shows release profiles ofmyoglobin encapsulated to PLGA (with the end group blocked) microsphereswith and without protection of AqueSpheres. When myoglobin wasencapsulated as pure protein particles into microspheres made ofester-end PLGA, only 17% of the loaded protein was released over 45days. For myoglobins encapsulated after pre-loaded in AqueSpheres, up to75% of the loaded protein was released linearly over 45 days without aburst release in the beginning. Such a burst-free linear release wasalso achieved when the myoglobin-dextran particles were encapsulated inmicrospheres of a relatively hydrophilic acid-ended PLGA (FIG. 12).

FIG. 12. shows the myoglobin release profiles from microspheres made ofacid-end PLGA (molecular weight=12K) with lactide:glycolide ratio of50:50, 65:35 and 75:25, respectively. For all these samples, myoglobinwere pre-formulated to AqueSpheres prior to encapsulation into PLGAmicrospheres. About 7 to 12% loadings were released in the first day,followed by a linear kinetics. From microspheres made of PLGA with L/Gof 50/50 and 65/35 and MW of 12K, protein release was over 90% in 50days, almost complete. Increase in the L/G ratio from 65/35 to 75/25resulted in slightly a decreased release rate as that 80% of loadingswas released in the same time period. Release rate also declined byincrease of molecular weight (MW) from 12K to 20K. For the PLGA with L/Gratio of 65/35, 65% of myoglobin encapsulated was released during 50days. In either of the cases, the release profile were almost linear.Encapsulation efficiency of myoglobin into PLGA microspheres by thismethods was about 90% based on analysis of the protein content in thesupernatant of after the preparation process.

Example 10 Bioactivity of GM-CSF Released from PLGA Microsphers

The protein, rhGM-CSF was loaded into PLGA microspheres throughAqueSpheres as the methods described in Example 1, 2 and 3. The proteinto dextran ratio was 1:500 and the AqueSphere to PLGA ratio was 1:5. TherhGM-CSF loaded PLGA microspheres were suspended in a buffer solutionand incubated at 37° C. The supernatant was collected each day andreplaced by fresh buffer. The collected supernatant was diluted by 20times and assayed as in Example 7. The activities measured are plottedagainst the sampling dates in FIG. 13. The activity was roughly constantup to day 24 after incubation, then dropped to the level of negativecontrol at day 32.

It has been widely recognized that local acidity generated inside of thePLGA microsphres due to the polymer degradation is one of the majorcause for protein denature during the release period {26}. To examinethe effect of scidity on the activity of rhGM-CSF, the protein wasincubated in dextran solutions at pH of 1, 2, 3, 4, 5 and 6,respectively, for one day prior to activity assay. Compared with thesample incubated at pH 6, the activity reduced by 75% at pH 4, andreduced to 45% when the pH was below 2. This pH dependent activitydeclining was not observed for the protein released from the PLGAmicrospheres (FIG. 13). This result suggests that local acidity was notaccumulated in the matrix of the PLGA microspheres. Probably AqueSpheresformed viscouse channels upon hydration which is, although lesspermeable to macromolecular agents, highly permeable to small molecularbuffer so that the acidic group generated by PLGA degradation werebuffered during the protein release period.

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1. A method for encapsulating agents into particles through stableaqueous-aqueous emulsification comprising: a. selecting polysaccharidesas the dispersed phase for aqueous-aqueous emulsification, selectingaqueous polymers as the continuous phase, and selecting an stabilizingagent and its concentration for aqueous-aqueous emulsification, toprovide a stable polymer aqueous-aqueous emulsion which is capable ofencapsulating an agent into the polysaccharide dispersed phase; b.providing at least one agent; c. controlling the size and shape of theagent-loaded polysaccharide particles into appropriate size range; d.drying the emulsion; and e. removing the continuous phase after dryingby washing the sample with solvent(s) which do not penetrate into thedried dispersed phase nor affect the loaded delicate agent(s).
 2. Acomposition used in the method of claim 1, including an aqueousdispersed phase, an aqueous continuous phase and an aqueous surfacemodifier, capable to form a stable aqueous-aqueous emulsion.
 3. Thecomposition of claim 2, comprising sufficient amount of polysaccharidesor derivatives thereof capable of forming the dispersed phase of theaqueous-aqueous emulsion and protecting agents encapsulated.
 4. Thecomposition of claim 3, wherein the polysaccharide is selected from thegroup consisting of dextran, starch, cellulose and its derivatives, andagarose and all type of poly- or oligo-sugars, which possess similarstructure.
 5. The composition of claim 4, wherein the average molecularweight of the polysaccharides is ranged from 2,000 to 2,000,000.
 6. Thecomposition of claim 3, wherein the agent is a biologically activeagent.
 7. The composition of claim 6, wherein the agent is selected fromthe group consisting of proteins, peptides, DNA/RNA, liposomes, and liveviruses.
 8. The composition of claim 7, wherein the protein or peptideis selected from the group consisting of erythropoietin (EPO),granulocyte colony stimulating factor (G-CSF), granulocyte macrophagecolony stimulating factor (GM-CSF), interferon and β, growth hormone,calcitonin, tissue-type plasminogen activator (TPA), factor VIII, factorIX, hirudin, dornabe, and other therapeutic proteins or peptides.
 9. Thecomposition of claim 3, further comprising a small molecular sugar ascomplimentary agents for better protection of agents encapsulated in thepolysaccharide dispersed phase during successive steps.
 10. Thecomposition of claim 9, wherein the small molecular sugar is selectedfrom trehalose, manitol, sucrose, lactose or glycerin.
 11. Thecomposition of claim 2, comprising an aqueous polymer, which isimmiscible with the polysaccharides, to form the continuous phase of theaqueous-aqueous emulsion.
 12. The composition of claim 11, wherein theaqueous polymer in the continuous phase is polyethylene glycol (PEG),polyethylene oxide (PEO), polyvinyl pyrrolidone (PVP), or polyvinylalcohol (PVA).
 13. The composition of claim 12, wherein the averagemolecular weight of the polymer is ranged from 2,000 to 2,000,000. 14.The composition of claim 2, comprising an aqueous polymer as the surfacemodifier of the dispersed phase.
 15. The composition of claim 14,wherein the polymeric surface modifier is selected from sodium alginate,hyaluronate, carboxymethyl cellulose, carboxymethyl dextran, dextransulfate, and other dextran or starch devertives, or other polymers thatpossess negatively charged backbone and positively charged counter ions.16. The method of claim 1, wherein the emulsion is dried throughlyophilization, spray drying or a conventional drying process tosolidify the agent-encapsulated polysaccharide dispersed phase. 17.Dried polysaccharide dispersed phase prepared by the method of claim 16,possessing an average diameter of 1-5 μm for inhalation and for doublemicroencapsulation, and of 1-50 μm for other applications.
 18. A methodof encapsulating dried polysaccharide dispersed phase into biodegradablepolymer microspheres for controlled release of bioactive agent(s)comprising: a) utilizing a solid-in-oil-in-water (S-O-W) emulsificationprocess or a solid-in-oil-in-oil process with the dried polysaccharidedispersed phase as the solid phase; b) selecting a biodegradablepolymer, dissolving the polymer in an organic solvent and suspending thedried polysaccharide dispersed phase in the polymer solution; c)selecting polymeric surfactant(s) for dispersing the solution of thebiodegradable polymers in a water solution of a small molecular salt; d)the concentration of the slat solution ranges from 0.5% to 50%; e)removing the organic solvent by extraction or evaporation.
 19. Themethod of claim 18, wherein the biodegradable polymer is PLGA,poly-pseudo CBZ-serine or other polymers.
 20. Particulates of degradablepolymers prepared using the method of claim 18, wherein driedpolysaccharide dispersed phase is distributed in the matrix. 21.Particulates of claim 20, wherein the ratio of dried polysaccharidedispersed phase to the degradable polymer is within the range of 1:2 to1:40.
 22. A composition of any one of claims 2-15 for or acceptable forpharmaceutical applications.